Ruixia Ma

CRISPR/cas9, a novel genomic tool to knock down microRNA in vitro and in vivo.

MicroRNAs are small and non-coding RNA molecules with the master role in regulation of gene expression at post-transcriptional/translational levels. Many methods have been developed for microRNA loss-of-function study, such as antisense inhibitors and sponges; however, the robustness, specificity, and stability of these traditional strategies are not highly satisfied. CRISPR/cas9 system is emerging as a novel genome editing tool in biology/medicine research, but its indication in microRNA research has not been studied exclusively. In this study, we clone CRISPR/cas9 constructs with single-guide RNAs specifically targeting biogenesis processing sites of selected microRNAs; and we find that CRISPR/cas9 can robustly and specifically reduce the expression of these microRNAs up to 96%. CRISPR/cas9 also shows an exclusive benefit in control of crossing off-target effect on microRNAs in the same family or with highly conserved sequences. More significantly, for the first time, we demonstrate the long term stability of microRNA knockdown phenotype by CRISPR/cas9 in both in vitro and in vivo models.

Inhibition of breast cancer cell motility with a non-cyclooxygenase inhibitory derivative of sulindac by suppressing TGFβ/miR-21 signaling

Compelling efficacy on intervention of tumorigenesis by nonsteroidal anti-inflammatory drugs (NSAIDs) has been documented intensively. However, the toxicities related to cyclooxygenase (COX) inhibition resulting in suppression of physiologically important prostaglandins limit their clinical use for human cancer chemoprevention. A novel derivative of the NSAID sulindac sulfide (SS), referred as sulindac sulfide amide (SSA), was recently developed, which lacks COX inhibitory activity, yet shows greater suppressive effect than SS on growth of various cancer cells. In this study, we focus on the inhibitory activity of SSA on breast tumor cell motility, which has not been studied previously. Our results show that SSA treatment at non-cytotoxic concentrations can specifically reduce breast tumor cell motility without influencing tumor cell growth, and the mechanism of action involves the suppression of TGFβ signaling by directly blocking Smad2/3 phosphorylation. Moreover, miR-21, a well-documented oncogenic miRNA for promoting tumor cell metastasis, was also found to be involved in inhibitory activity of SSA in breast tumor cell motility through the modulation of TGFβ pathway. In conclusion, we demonstrate that a non-COX inhibitory derivative of sulindac can inhibit breast tumor metastasis by a mechanism involving the TGFβ/miR-21 signaling axis.

Abstract 4790: Let-7 mediates sulindac inhibition of tumor cell transformation

Cell transformation is the process in which normal cells acquire the properties of cancer. Sulindac Sulfide (SS) is one of non-steroidal anti-inflammatory drugs (NSAIDs), and its anticancer activity in prevention of tumor occurrence and progression has been documented in various human cancers. In this study, we observed the anti-transformative activity of SS in NIH/3T3 cells by using a two-stage transformation assay induced by the carcinogens, 3-methylcholanthrene (MCA) and 2-O-tetradecanoylphorbol-13-acetate (TPA) in which foci formation is used to characterize the transformed cells. We found that SS could inhibit foci formation by attenuation of TPA’s promotive effect. K-Ras was upregulated in the transformed cells, while SS treatment could selectively inhibit the growth of transformed cell in which K-Ras was suppressed significantly. Loss-of-function study demonstrated that knockdown of K-Ras in NIH/3T3 cells could significantly reduce foci formation, which support that K-Ras might be one of the key targets involved in SS inhibiting tumor cell transformation. When studying the mechanism by which SS regulates K-Ras, we triggered tumor suppressor miRNA, let-7b, which was previously reported to target K-Ras and repress its expression through the post-transcriptional or translational modulation. We found that let-7b could be upregulated in the transformed cells by SS treatment. However, the inhibitory effect of SS on foci formation was significantly attenuated when let-7b was knocked down by CRISPR/Cas9. In parallel, K-Ras maintained static expression in the transformed cells, regardless of SS treatment. Meanwhile, the overexpression of endogenous let-7b can significantly inhibit tumor transformation. In addition, we demonstrated that SS could suppress the expression of Lin28 that interacts with let-7 through a documented feedback loop. Given our previous studies reporting that SS was able to inhibit the transcriptional activity of NF-κB that is involved in regulation of Lin28 expression, our study demonstrates that the NF-κB/Lin28/Let-7/K-Ras axis addresses a new mechanism responsible for anti-transformative activity of SS. These results will provide novel insights into understanding the mechanisms involved in tumorigenesis and benefiting identification of new targets for drug development in prevention of tumor progression. Citation Format: Zhipin Liang, Bin Yi, Hongyou Zhao, Ruixia Ma, Yaguang Xi. Let-7 mediates sulindac inhibition of tumor cell transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4790. doi:10.1158/1538-7445.AM2017-4790

Abstract B26: MiR-200 is involved in anti-invasive activity of sulindac in colon cancer

The chemopreventive benefits of nonsteroidal anti-inflammatory drugs (NSAIDs) are documented in various human cancers by numerous pre-clinical and clinical studies. However, the mechanisms accounting for NSAIDs9 anticancer activity have not been well understood, given the controversial findings when determining the key role of cyclooxygenase 2 (COX-2) inhibition in the action. MicroRNA is a set of non-coding small RNA molecules showing the “master” role in regulation of human coding gene expression. We are interested in studying if microRNA is able to address the underlying mechanism of NSAIDs9 anticancer activity. MiR-200 is a tumor suppressor microRNA and is well-known for its inhibitory effect on the epithelial-mesenchymal transition (EMT). Our previous study reported that the NSAID, sulidac sulfide (SS) can inhibit breast and colon tumor cell motility through a distinct mechanism from the known suppressive activity on tumor cell growth through COX-2 inhibition. In this study, we aim to study if miR-200 is involved in this underlying mechanism. By using human colon tumor cell lines, HCT116 and LIM2405, as the research models, we found that SS treatment could ultimately suppress the tumor cell invasion, while miR-200 was upregulated in parallel. When examining the expression levels of a panel of EMT related genes, we found the E-cadherin was upregulated but snail was downregulated inversely. E-cadherin was reported to be regulated by snail through the transcriptional repression; however, our results did not support the direct control of snail on E-cadherin in the colon tumor cells with miR-200 expression. When snail and miR-200 were forced to be overexpressed in HCT116 cells, E-cadherin was upregulated; when snail was knocked down by siRNAs in miR-200 overexpressed cells, E-cadherin maintained the static expression. These results suggest that the regulation of snail on E-cadherin in colon cancer cells may depend on miR-200 expression. To further support this hypothesis, we studied the interaction between snail and miR-200. The bioinformatics analysis suggested that several E-boxes on miR-200 gene promoter could serve as the binding sites of snail. By employing the chromatin immunoprecipitation (ChIP) and luciferase assays, we demonstrated that snail could directly bind to miR-200 promoter and regulate its expression at the transcriptional level. Our previous study showed that blockade NF-κB signaling is involved in SS inhibition of colon tumor cell motility. Given that snail was also reported to be regulated by NF-κB through the transcriptional modulation, these data suggest the NF-κB-snail-miR-200-E-cadherin axis may account for a novel mechanism to address the anti-metastatic activity of sulindac in colon cancer. Potentially, our results will provide insights into development of novel drugs in treatment of cancer patients with advanced diseases. This study is supported by the NIH/NCI R01 Grant (1R01CA192395) and the American Cancer Society Research Scholar Grant (RSG-13-265-01-RMC). Citation Format: Hong Chang, Xiangling Feng, Ruixia Ma, Yaguang Xi. MiR-200 is involved in anti-invasive activity of sulindac in colon cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B26.

Abstract B32: Oncogenic role of snoRD93 in breast cancer cells

Noncoding RNAs (ncRNAs) play key roles in many essential processes of human diseases through regulating gene expression. Small nucleolar RNA (snoRNA) is a relatively large group of ncRNAs with diversified functions in mammalian cells, although many of them have not been well-characterized. Recent studies have revealed the alterations of different snoRNAs in prostate, breast, and lung malignancies; however, their mechanistic roles in tumorigenesis and progression have not been well-studied yet. In this study, we compared the transcriptome profiles of human breast cancer cell lines, MCF-7 and MDA-MB-231, by using the next generation sequencing (NGS). MCF-7 cells were derived from the primary breast tumor, while MDA-MB-231 cells are highly metastatic. Our sequencing results showed that 13 snoRNAs was significantly overexpressed in MDA-MB-231 cells versus MCF-7 cells. In particular, we are interested in snoRD93, given its expression showing 27-time higher in MDA-MB-231 cells than MCF-7 cells. We further studied the oncogenic role of snoRD93 in MDA-MB-231 cells by using loss-of-function strategy. By employing cell viability assay, we found that the repression of snoRD93 by transfection of the antisense inhibitors could significantly inhibit the tumor cell growth. In addition, downregulation of snoRD93 could result in relatively low motility of MDA-MB-231 cells as shown in the matrigel invasion assay. Therefore, our data support the oncogenic role of snoRD93 in breast cancer cells and suggest that snoRD93 can be triggered a potential target for development of new anticancer drugs. Citation Format: Ruixia Ma, Hongyou Zhao, Dillon Patterson, Glen Borchert, Yaguang Xi. Oncogenic role of snoRD93 in breast cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B32.

Abstract B34: Inhibition of breast cancer cell metastasis with a non-cyclooxygenase inhibitory derivative of sulindac by suppressing TGFbeta/miR-21 signaling

Compelling efficacy on intervention of tumor occurrence and progression by nonsteroidal anti-inflammatory drugs (NSAIDs) has been intensively reported in numerous preclinical and clinical studies. However, the toxicities related to cyclooxygenase (COX) inhibition resulting in suppression of physiologically important prostaglandins limit their clinical use for human cancer chemoprevention. A novel derivative of the NSAID, sulindac sulfide (SS), referred as sulindac sulfide amide (SSA) was recently developed, which lacks COX inhibitory activity, yet shows greater suppressive effect than SS on growth of variable cancer cells. In this study, we focus on the inhibitory activity of SSA on breast tumor cell motility, which has not been studied previously. Our results showed that SSA could inhibit breast tumor cell invasion and migration at sub-cytotoxic concentrations; whereas we previously reported that SS has similar activity on breast and colon tumor cells but at a concentration (50 µM) over 10 times higher than SSA (4 µM). We found that miR-21, a well-documented oncogenic miRNA for promoting tumor cell metastasis, could be downregulated by SSA and involved in mediation of anti-invasive activity of SSA in breast tumor cells. When studying the mechanisms responsible for the regulation of miR-21 by SSA, we demonstrated that TGFbeta signaling suppressed by SSA could ultimately result in downregulation of miR-21 through the transcriptional control. Reduction of Smad2 and Smad3 phosphorylation was the direct response to SSA treatment in metastatic breast tumor MDA-MB-231 cells. By using ChIP assay, we demonstrated that phosphorylated Smad2/3 could bind to the promoter of miR-21 gene. Therefore, in this project, we demonstrate that a non-COX inhibitory derivative of sulindac can inhibit breast tumor metastasis through a new mechanism involving the TGFbeta/miR-21 signaling axis. This study is supported by the NIH/NCI R01 Grant (1R01CA192395) and the American Cancer Society Research Scholar Grant (RSG-13-265-01-RMC). Citation Format: Bin Yi, Hong Chang, Xiangling Feng, Ruixia Ma, Gary A Piazza, Yaguang Xi. Inhibition of breast cancer cell metastasis with a non-cyclooxygenase inhibitory derivative of sulindac by suppressing TGFbeta/miR-21 signaling. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B34.

Abstract B31: Sulindac inhibition of tumor cell transformation

Cell transformation is the process in which normal cells acquire the properties of cancer. Sulindac Sulfide (SS) is one of non-steroidal anti-inflammatory drugs (NSAIDs), and its anticancer activity in prevention of tumor occurrence and progression has been documented in various human cancers. In this study, we observed the anti-transformative activity of SS in NIH/3T3 cells by using a two-stage transformation assay induced by the carcinogens, 3-methylcholanthrene (MCA) and 2-O-tetradecanoylphorbol-13-acetate (TPA) in which foci formation is used to characterize the transformed cells. We found that SS could inhibit foci formation by attenuation of TPA9s promotive effect. K-Ras was upregulated in the transformed cells, while SS treatment could selectively inhibit the growth of transformed cell in which K-Ras was suppressed significantly. Loss-of-function study demonstrated that knockdown of K-Ras in NIH/3T3 cells could significantly reduce foci formation, which support that K-Ras might be one of the key targets involved in SS inhibiting tumor cell transformation. When studying the mechanism by which SS regulates K-Ras, we triggered two tumor suppressor miRNAs, let-7b and let-7g, which were previously reported to target K-Ras and repress its expression through the post-transcriptional or translational modulation. We found that let-7b and let-7g could be upregulated in the transformed cells by SS treatment. However, the inhibitory effect of SS on foci formation was significantly attenuated when let-7b and let-7g were knocked down by CRISPR/cas9. In parallel, K-Ras maintained static expression in the transformed cells, regardless of SS treatment. These results suggest that upregulation of let-7b and let-7g is the key step for SS to inhibit tumor transformation. In addition, we demonstrated that SS could suppress the expression of Lin28 that interacts with let-7 through a documented feedback loop. Given our previous studies reporting that SS was able to inhibit the transcriptional activity of NF-kappaB that is involved in regulation of Lin28 expression, our study demonstrates that the NF-kappaB/Lin28/Let-7/K-Ras axis addresses a new mechanism responsible for anti-transformative activity of SS. These results will provide novel insights into understanding the mechanisms involved in tumorigenesis and benefiting identification of new targets for drug development in prevention of tumor progression. This study is supported by the NIH/NCI R01 Grant (1R01CA192395) and the American Cancer Society Research Scholar Grant (RSG-13-265-01-RMC). Citation Format: Zhipin Liang, Xiangling Feng, Hong Chang, Bin Yi, Ruixia Ma, Yaguang Xi. Sulindac inhibition of tumor cell transformation. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B31.

Abstract 1965: CRISPR/cas9, an innovative genomic tool to knock down microRNA

MicroRNAs are a set of small and non-coding RNA molecules showing the “master” role in regulation of coding-gene expression at the post-transcriptional/translational levels. Several methods have been developed for microRNA loss-of-function study, such as antisense inhibitors and sponges; however, their robustness, specificity, and stability cannot be highly satisfied with the increased demand on microRNA functional research. CRISPR/cas9 system is emerging as a novel genome editing tool that has been exclusively studied in coding-genes knockdown, but its indication in microRNA research has not been explored yet. In this project, we clone CRISPR/cas9 constructs with designated single guide RNAs targeting biogenesis processing sites of the selected microRNAs, miR-200b and miR-17. Our results demonstrate that CRISPR/cas9 can robustly reduce the expression of these microRNAs up to 96%. More significantly, our data support high specificity and low off-target effect of CRISPR/cas9 in editing microRNA genes. Given this feature, it possesses an advantage to knockdown microRNAs in same family or with highly conserved sequences superior to antisense inhibitors and sponges. In addition, for the first time, we demonstrate the long term stability of microRNA knockdown phenotype by CRISPR/cas9 in both in vitro and in vivo models. In conclusion, we develop a novel method by adapting CRISPR/cas9 system to downregulate microRNA expression, which shows improved robustness, specificity, and stability than the previously established methodologies. Citation Format: Hong Chang, Bin Yi, Ruixia Ma, Hongyou Zhao, Yaguang Xi. CRISPR/cas9, an innovative genomic tool to knock down microRNA. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1965.

Abstract 4125: SSA, a novel sulindac derivative, inhibits breast cancer cell invasion and migration

Nonsteroidal anti-inflammatory drugs (NSAIDs), such as sulindac, have been reported for striking chemopreventive activity in various types of human cancer, including breast cancer. However, few studies have reported that sulindac is able to prevent tumor metastasis. In this study, we aim to study anti-metastatic activity of sulindac in breast cancer and explore the mechanistic basis responsible for this activity, which we hypothesize may involve a cyclooxygenase (COX) independent mechanism. Numerous studies have reported that the anticancer properties of sulindac might involve, at least partially, a non-COX mediated mechanism, which provides an insight into development of safer and more efficacious new derivatives. We have previously described a chemical derivative of sulindac, referred to as sulindac sulfide amide (SSA), which lacks inhibitory effect on COX-1 or -2, yet shows greater potency to inhibit tumor cell growth compared with sulindac sulfide (SS). Our results showed that SSA treatment at a sub-cytotoxic concentration (4μM) for 36 h significantly inhibits both migration and invasion of highly aggressive MDA-MB-231, BT-20 and SK-BR-3 breast tumor cells. When treating non-invasive breast MCF-7 cells with TGF-β1 to induce their motility, we found that SSA could significantly counteract the induced motility of these cells. These results indicate that SSA can suppress breast tumor cell invasion by modulation of TGF-β1 signaling. We further studied Smad-2/3 that are important downstream components in the TGF-β1 signaling pathway and found that SSA could inhibit Smad-2/3 phosphorylation and reduce their translocation to the nucleus. In addition, Smad-2/3 knockdown could mimic the anti-invasive effect of SSA in MDA-MB-231 cells, regardless of the presence of TGF-β1. In summary, we demonstrate that the non-COX inhibitory derivative, SSA can suppress breast cancer cell invasion and migration. These observations suggest that inhibition of TGF-β1/Smad signaling is responsible for the inhibitory effect of SSA on breast tumor cell invasion and migration. Citation Format: Bin Yi, Xingling Feng, Ruixia Ma, Xiaoguo Zhang, Hong Chang, Hongyou Zhao, Ziping Liang, Xi Chen, Xiuhua Hu, Gary Piazza, Yaguang Xi. SSA, a novel sulindac derivative, inhibits breast cancer cell invasion and migration. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4125. doi:10.1158/1538-7445.AM2015-4125

Abstract P1-07-25: SSA, a novel sulindac sulfide derivative, inhibits tumor cell growth and invasion in breast cancer

Nonsteroidal anti-inflammatory drugs (NSAIDs), such as sulindac sulfide (SS), have been reported for striking chemopreventive activities in various types of human malignances, including breast cancer. However, the toxicities related to cyclooxygenase (COX) inhibition resulting in suppression of physiologically important prostaglandins limit their clinical use for chemoprevention in human cancer. We recently developed a novel amine derivative of SS named as SSA, which lacks inhibitory effect on COX-1 or -2, yet shows 10 times greater suppressive effect than SS on growth of a panel of breast cells. Moreover, SSA treatment at sub-cytotoxic concentration (4μM) for 36 hr can significantly inhibit both migration and invasion of highly aggressive breast tumor MDA-MB-231cells. To understand the molecular mechanism accounting for this activity, we examined 4 oncogenic miRNAs, including mir-17-92, mir-9, mir-10b, and mir-21 that were previously reported by our group to be associated with SS anti-invasive activity in breast and colorectal cancer. We found SSA could significantly down-regulate these miRNAs; whereas their forced expression was able to counteract the anti-invasive activity of SSA in MDA-MB-231 cells. There results imply that significance of SSA with non-COX inhibitory properties in suppression of tumor cell invasion could provide novel insights into development of safer and more effective strategies for prevention of breast cancer progression and metastasis. In addition, after inducing mobility of non-invasive breast MCF-7 cells by using TGF-β1, we treated these invading cells with SSA and found that their mobility was significantly decreased. These results support anti-invasive activity of SSA in human breast cancer cells and inhibition of TGF-β1 as well as oncogenic miRNAs may be responsible for the mechanistic basis by which SSA prevents breast tumor cell invasion. This study is supported by an American Cancer Society Research Scholar Grant (RSG-13-265-01-RMC) and NIH/NCI R21 Grants (5R21CA160280 and 1R21CA182754) to Yaguang Xi. Citation Format: Bin Yi, Xiangling Feng, Hong Chang, Ruixia Ma, Xiaoguo Zhang, Gary A Piazza, Yaguang Xi. SSA, a novel sulindac sulfide derivative, inhibits tumor cell growth and invasion in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-25.